The Vid vesicle to vacuole trafficking event requires components of the SNARE membrane fusion machinery

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is targeted to Vid vesicles when glucose-starved cells are replenished with glucose. Vid vesicles then deliver FBPase to the vacuole for degradation. A modified alkaline phosphatase assay was developed to study the trafficking of Vid vesicles to the vacuole. For this assay, FBPase was fused with a truncated form of alkaline phosphatase. Under in vivo conditions, FBPase-delta60Pho8p was targeted to the vacuole via Vid vesicles, and it exhibited Pep4p- and Vid24p-dependent alkaline phosphatase activation. Vid vesicle-vacuole targeting was reconstituted using Vid vesicles that contained FBPase-delta60Pho8p. These vesicles were incubated with vacuoles in the presence of cytosol and an ATP-regenerating system. Under in vitro conditions, alkaline phosphatase was also activated in a Pep4p- and Vid24p-dependent manner. The GTPase Ypt7p was identified as an essential component in Vid vesicle-vacuole trafficking. Likewise, a number of v-SNAREs (Ykt6p, Nyv1p, Vti1p) and homotypic fusion vacuole protein sorting complex family members (Vps39p and Vps41p) were required for the proper function of Vid vesicles. In contrast, the t-SNARE Vam3p was a necessary vacuolar component. Vid vesicle-vacuole trafficking exhibits characteristics similar to heterotypic membrane fusion events.     

Contrasting selection modes at the Adh1 locus in outcrossing Miscanthus sinensis vs. inbreeding Miscanthus condensatus (Poaceae)

We estimated DNA sequence variation of the Adh1 locus in the outcrossing Miscanthus sinensis (Poaceae) and its close selfing relative, M. condensatus. Tajima's test of selection is significantly negative for both overall exons and replacement sites in M. sinensis. Among its entire sample, nucleotide diversity of nonsynonymous sites is higher than that of synonymous sites. A McDonald and Kreitman test of neutrality indicates an excess of intraspecific replacement polymorphisms, suggesting possible directional selection toward advantageous mutants. However, frequent intragenic recombination suggests both purifying and positive selection is unlikely. Recent demographic expansions coupled with relaxation of purifying selection may have resulted in elevated genetic diversity at the Adh1 locus as well as the trnL-trnF intergenic spacer of cpDNA in this outcrossing species. In contrast, low levels of genetic diversity were detected at both the Adh1 locus and the cpDNA spacer in M. condensatus, consistent with bottlenecks associated with selfing in all populations. While Tajima's D and Fu and Li's F statistics did not reveal deviation from neutrality at the Adh1 locus in M. condensatus, 12 replacements vs. 10 synonymous changes were detected. Based on pairwise comparisons of the d(N)/d(S) ratio, lineages of closely related populations of the species distributed along saline habitats appeared to be under directional selection.     

Testing of the additivity-based protein sequence to reactivity algorithm

The standard free energies of association (or equilibrium constants) are predicted for 11 multiple variants of the turkey ovomucoid third domain, a member of the Kazal family of protein inhibitors, each interacting with six selected enzymes. The equilibrium constants for 38 of 66 possible interactions are strong enough to measure, and for these, the predicted and measured free energies are compared, thus providing an additional test of the additivity-based sequence to reactivity algorithm. The test appears to be unbiased as the 11 variants were designed a decade ago to study furin inhibition and the specificity of furin differs greatly from the specificities of our six target enzymes. As the contact regions of these inhibitors are highly positive, nonadditivity was expected. Of the 11 variants, one does not satisfy the restriction that either P(2) Thr or P(1)' Glu should be present and all three measurable results on it, as expected, are nonadditive. For the remaining 35 measurements, 22 are additive, 12 are partially additive, and only one is (slightly) nonadditive. These results are comparable to those obtained for a set of 398 equilibrium constants for natural variants of ovomucoid third domains. The expectation that clustering of charges would be nonadditive is modified to the expectation that major nonadditivity will be observed only if the combining sites in both associating proteins involve large charge clusters of the opposite sign. It is also shown here that an analysis of a small variant set can be accomplished with a smaller subset, in this case 13 variants, rather than the whole set of 191 members used for the complete algorithm.     

Organelle DNA phylogeography of Cycas taitungensis, a relict species in Taiwan

The phylogegraphic pattern of Cycas taitungensis, an endemic species with two remaining populations in Taiwan, was investigated based on genetic variability and phylogeny of the atpB-rbcL noncoding spacer of chloroplast DNA (cpDNA) and the ribosomal DNA (rDNA) internal transcribed spacer (ITS) of mitochondrial DNA (mtDNA). High levels of genetic variation at both organelle loci, due to frequent intramolecular recombination, and low levels of genetic differentiation were detected in the relict gymnosperm. The apportionment of genetic variation within and between populations agreed with a migrant-pool model, which describes a migratory pattern with colonists recruited from a random sample of earlier existing populations. Phylogenies obtained from cpDNA and mtDNA were discordant according to neighbour-joining analyses. In total four chlorotypes (clades I-IV) and five mitotypes (clades A-E) were identified based on minimum spanning networks of each locus. Significant linkage disequilibrium in mitotype-chlorotype associations excluded the possibility of the recurrent homoplasious mutations as the major force causing phylogenetic inconsistency. The most abundant chlorotype I was associated with all mitotypes and the most abundant mitotype C with all chlorotypes; no combinations of rare mitotypes with rare chlorotypes were found. According to nested clade analyses, such nonrandom associations may be ascribed to relative ages among alleles associated with the geological history through which cycads evolved. Nested in networks as interior nodes coupled with wide geographical distribution, the most dominant cytotypes of CI and EI may represent ancestral haplotypes of C. taitungensis with a possible long existence prior to the Pleistocene glacial maximum. In contrast, rare chlorotypes and mitotypes with restricted and patchy distribution may have relatively recent origins. Newly evolved genetic elements of mtDNA, with a low frequency, were likely to be associated with the dominant chlorotype, and vice versa, resulting in the nonrandom mitotype-chlorotype associations. Paraphyly of CI and EI cytotypes, leading to the low level of genetic differentiation between cycad populations, indicated a short period for isolation, which allowed low possibilities of the attainment of coalescence at polymorphic ancestral alleles.     

Phylogeography of Kandelia candel in East Asiatic mangroves based on nucleotide variation of chloroplast and mitochondrial DNAs

Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.     

Free anterolateral thigh flap for extremity reconstruction: clinical experience and functional assessment of donor site

From August of 1995 through July of 1998, 38 free anterolateral thigh flaps were transferred to reconstruct soft-tissue defects. The overall success rate was 97 percent. Among 38 anterolateral thigh flaps, four were elevated as cutaneous flaps based on the septocutaneous perforators. The other 34 were harvested as myocutaneous flaps including a cuff of vastus lateralis muscle (15 to 40 cm3), either because of bulk requirements (33 cases) or because of the absence of a septocutaneous perforator (one case). However, vastus lateralis muscle is the largest compartment of the quadriceps, which is the prime extensor of the knee. Losing a portion of the vastus lateralis muscle may affect knee stability. Objective functional assessments of the donor sites were performed at least 6 months postoperatively in 20 patients who had a cuff of vastus lateralis muscle incorporated as part of the myocutaneous flap; assessments were made using a kinetic communicator machine. The isometric power test of the ratios of quadriceps muscle at 30 and 60 degrees of flexion between donor and normal thighs revealed no significant difference (p > 0.05). The isokinetic peak torque ratio of the quadriceps and hamstring muscles, including concentric and eccentric contraction tests, showed no significant difference (p > 0.05), except the concentric contraction test of the quadriceps muscle, which revealed mild weakness of the donor thigh (p < 0.05). In summary, the functional impairment of the donor thighs was minimal after free anterolateral thigh myocutaneous flap transfer.     

Sonic hedgehog signaling from the urethral epithelium controls external genital development

External genital development begins with formation of paired genital swellings, which develop into the genital tubercle. Proximodistal outgrowth and axial patterning of the genital tubercle are coordinated to give rise to the penis or clitoris. The genital tubercle consists of lateral plate mesoderm, surface ectoderm, and endodermal urethral epithelium derived from the urogenital sinus. We have investigated the molecular control of external genital development in the mouse embryo. Previous work has shown that the genital tubercle has polarizing activity, but the precise location of this activity within the tubercle is unknown. We reasoned that if the tubercle itself is patterned by a specialized signaling region, then polarizing activity may be restricted to a subset of cells. Transplantation of urethral epithelium, but not genital mesenchyme, to chick limbs results in mirror-image duplication of the digits. Moreover, when grafted to chick limbs, the urethral plate orchestrates morphogenetic movements normally associated with external genital development. Signaling activity is therefore restricted to urethral plate cells. Before and during normal genital tubercle outgrowth, urethral plate epithelium expresses Sonic hedgehog (Shh). In mice with a targeted deletion of Shh, external genitalia are absent. Genital swellings are initiated, but outgrowth is not maintained. In the absence of Shh signaling, Fgf8, Bmp2, Bmp4, Fgf10, and Wnt5a are downregulated, and apoptosis is enhanced in the genitalia. These results identify the urethral epithelium as a signaling center of the genital tubercle, and demonstrate that Shh from the urethral epithelium is required for outgrowth, patterning, and cell survival in the developing external genitalia.     

Neuronal correlates of kinematics-to-dynamics transformation in the supplementary motor area

It is widely acknowledged that movements are planned at the level of the kinematics. However, the central nervous system must ultimately transform kinematic plans into dynamics-related commands. How, when, and where the kinematics-to-dynamics (KD) transformation is processed represent fundamental and unanswered questions. We recorded from the supplementary motor area (SMA) of two monkeys as they executed visually instructed reaching movements. We specifically analyzed a delay period following the instruction but prior to the go signal (motor planning). During the delay, a group of neurons in the SMA progressively came to reflect the dynamics rather than the desired kinematics of the upcoming movement. This finding suggests that some neurons in the SMA participate in the KD transformation.